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Abstract:

In general, the invention features substantially purified MANF and
substantially purified nucleic acids encoding the same. The invention
also features a pharmaceutical composition that includes MANF and a
pharmaceutically-acceptable excipient, methods for treatment of a
neurodegenerative disease, methods for improving dopaminergic neuronal
survival during or following cell transplantation, methods for production
of neurons for transplantation, and methods for identifying compounds
that modulate or mimic MANF's biological activity.

18. A method of treating a subject having a disease or disorder of the
nervous system comprising: providing an effective amount of the
pharmaceutical composition of claim 9 to the subject.

19. The method of claim 9, wherein the disease is Parkinson's disease.

20. The method of claim 9, wherein the subject is human.

21. A method of preventing dopaminergic neuronal cell death in a mammal
comprising: providing an effective amount of a pharmaceutical composition
comprising a substantially purified polypeptide comprising the sequence
of SEQ ID NO: 3 or 4 to the subject.

22. The method of claim 21, wherein the subject is human.

23. A method for increasing the survival of dopaminergic neurons,
comprising a. contacting the dopaminergic neurons with a composition
comprising a substantially purified polypeptide comprising the sequence
of SEQ ID NO: 3 or 4, wherein the polypeptide is present in an amount
capable of promoting survival of the dopaminergic neuron.

24. The method of claim 23, wherein the contacting is performed in vitro.

Description:

CROSS-REFERENCE

[0001] This application is a continuation application of U.S. application
Ser. No. 12/535,029, filed Aug. 4, 2009, which is a continuation of U.S.
application Ser. No. 10/102,265, filed Mar. 20, 2002, which claims
benefit of and priority to provisional U.S. Application No. 60/277,516,
filed Mar. 20, 2001, each of which is incorporated herein by reference in
their entirety and to which applications we claim priority under 35 USC
§§119, 120.

BACKGROUND OF THE INVENTION

[0002] The invention relates to compositions and methods for increasing
the survival of neurons.

[0003] The growth, survival, and differentiation of neurons in the
peripheral and central nervous systems (PNS and CNS, respectively) are
dependent, in part, on target-derived, paracrine, and autocrine
neurotrophic factors. Conversely, the lack of neurotrophic factors is
thought to play a role in the etiology of neurodegenerative diseases such
as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral
sclerosis (ALS or Lou Gehrig's disease). In neuronal cell cultures,
neurotrophic support is provided by co-culturing with astrocytes or by
providing conditioned medium (CM) prepared from astrocytes. Astrocytes of
ventral mesencephalic origin exert much greater efficacy in promoting the
survival of ventral, mesencephalic dopaminergic neurons, compared with
astrocytes from other regions of the CNS, such as the neostriatum and
cerebral cortex. In chronic, mesencephalic cultures of 21 days in vitro
(DIV) or longer, the percentage of dopaminergic neurons increases from
20% to 60%, coincident with proliferation of a monolayer of astrocytes.
In contrast, in conditions in which the proliferation of astrocytes was
inhibited, dopaminergic, but not GABAergic neurons, were almost
eliminated from the cultures by 5 DIV. These results demonstrate the
importance of homotypically-derived astrocytes for the survival and
development of adjacent dopaminergic neurons, and suggest that
mesencephalic astrocytes are a likely source of a physiological,
paracrine neurotrophic factor for mesencephalic dopaminergic neurons.

[0004] The repeated demonstration that astrocytes secrete molecules that
promote neuronal survival has made astrocytes a focus in the search for
therapeutics to treat neurodegenerative diseases. Many laboratories have
attempted to isolate astrocyte-derived neurotrophic factors, but have
been hindered by a major technical problem: serum is an essential
component of the medium for the optimal growth of primary astrocytes in
culture, yet the presence of serum interferes with the subsequent
purification of factors secreted into the conditioned medium.

[0005] Thus, there is a need to identify and purify new neurotrophic
factors and to identify new methods to produce conditioned medium that
are compatible with protein isolation techniques.

SUMMARY OF THE INVENTION

[0006] We previously isolated a spontaneously immortalized type-1
astrocyte-like cell line, referred to as ventral mesencephalic cell
line-1 (VMCL-1). This cell line, deposited with the American Type Culture
Collection (ATCC; Manassas, Va.; ATCC Accession No: PTA-2479; deposit
date: Sep. 18, 2000), was derived from the ventral mesencephalon and
retained the characteristics of primary, type-1 astrocytes, but grows
robustly in a serum-free medium. The CM prepared from these cells
contains one or more neuronal survival factors that increase the survival
of mesencephalic dopaminergic neurons at least 3-fold, and promotes their
development as well.

[0007] Using a multi-step purification process, we have identified
arginine-rich protein (ARP) as a protein that co-purifies with the
dopaminergic neuronal survival-promoting activity of VMCL-1 CM. As the
protein and the activity co-purified through five purification steps, we
conclude that this protein is one of the factors in the VMCL-1 CM having
the desired dopaminergic neuronal survival-promoting activity.

[0008] We have also discovered that ARP is produced in a previously
unrecognized secreted form; we refer to this form as MANF (mature
astrocyte-derived neurotrophic factor). MANF lacks the N-terminal
arginine-rich portion of the protein, as is shown in FIG. 1 and SEQ ID
NO: 3. Based on examination of the sequences, we believe that this
secreted form results from the cleavage of a previously unidentified
splice variant of ARP (ARPβ or pro-MANF), which has the sequence
shown in SEQ ID NO: 2. MANF and pro-MANF, and biologically active
analogs, derivatives, and fragments thereof, are collectively referred to
as "MANF polypeptides."

[0009] Accordingly, in a first aspect, the invention features a
substantially purified MANF polypeptide. In one embodiment, the MANF
polypeptide has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or
7. In another embodiment, the MANF polypeptide includes one or more
conservative amino acid substitutions relative to the amino acid sequence
of SEQ ID NO: 2, 3, 4, 5, 6, or 7, or is otherwise substantially
identical to a protein having one of these amino acid sequences.

[0010] In a second aspect, the invention features a substantially purified
polynucleotide encoding a MANF polypeptide. As described above, the MANF
polypeptide may have the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6,
or 7, or may have one or more conservative amino acid substitutions
relative to these amino acid sequences. In one embodiment, the
polynucleotide encodes a protein substantially identical to a protein
having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7. In
another embodiment, the polynucleotide consists of the sequence of SEQ ID
NO: 9 or 10.

[0011] In a third aspect, the invention features an expression vector that
includes the polynucleotide of the second aspect. The expression vector
can be, for example, an adenoviral vector or a retroviral vector. In one
particular embodiment, the polynucleotide is operably linked to
regulatory sequences that allow for the expression of the polynucleotide
in a neural cell.

[0012] In a fourth aspect, the invention features a pharmaceutical
composition that includes: (i) a substantially purified MANF polypeptide;
and (ii) a carrier that is pharmaceutically acceptable for administration
to the central nervous system.

[0013] In a fifth aspect, the invention features a pharmaceutical
composition that includes: (i) a substantially purified MANF polypeptide;
(ii) a pharmaceutically acceptable carrier; and (iii) a neural cell. The
neural cell can be, for example, a neuron, a neural stem cell, or a
neuronal precursor cell.

[0014] In a sixth aspect, the invention features a method for increasing
survival of dopaminergic neurons, the method including the step of
contacting the dopaminergic neurons with a survival-promoting amount of a
substantially purified MANF polypeptide.

[0015] In a seventh aspect, the invention features a method for growing
dopaminergic neurons for transplantation. This method includes the step
of culturing the neurons, or progenitor cells thereof, with a
survival-promoting amount of a substantially purified MANF polypeptide.
In one embodiment, the MANF polypeptide is administered with a
pharmaceutically acceptable excipient.

[0016] In an eighth aspect, the invention features a method of treating a
patient having a disease or disorder of the nervous system. The method
including the step of administering to the patient a dopaminergic
neuronal survival-promoting amount of a substantially purified MANF
polypeptide.

[0017] In a ninth aspect, the invention features a method for preventing
dopaminergic neuronal cell death in a mammal. This method includes the
step of administering to the mammal a dopaminergic neuronal
survival-promoting amount of a substantially purified MANF polypeptide.

[0018] In a tenth aspect method of transplanting cells into the nervous
system of a mammal such as a human, including (i) transplanting cells
into the nervous system of the mammal; and (ii) administering a
dopaminergic neuronal survival-promoting amount of a MANF polypeptide to
the mammal in a time window from two to four hours before transplanting
the cells to two to four hours after transplanting the cells.

[0019] In an eleventh aspect, the invention features another method of
transplanting cells into the nervous system of a mammal such as a human.
This method includes the steps of: (a) contacting the cells with a MANF
polypeptide; and (b) transplanting the cells of step (a) into the nervous
system of the mammal. It is desirable that step (a) and step (b) be
performed within four hours of each other.

[0021] As demonstrated herein, dopaminergic neurons are, in large part,
prevented from dying in the presence of a MANF polypeptide. Dopaminergic
neurons of the mesencephalon die in patients having Parkinson's disease.
The invention thus provides a treatment of Parkinson's disease. In
addition, the use of a MANF polypeptide in the treatment of disorders or
diseases of the nervous system in which the loss of dopaminergic neurons
is present or anticipated is included in the invention.

[0022] The discovery that MANF is involved in dopaminergic neuronal
survival allows MANF to be used in a variety of diagnostic tests and
assays for identification of dopaminergic neuronal survival-promoting
drugs. MANF expression can also serve as a diagnostic tool for
determining whether a person is at risk for a neurodegenerative disorder.
This diagnostic process can lead to the tailoring of drug treatments
according to patient genotype (referred to as pharmacogenomics),
including prediction of the patient's response (e.g., increased or
decreased efficacy or undesired side effects upon administration of a
compound or drug).

[0023] Antibodies to a MANF polypeptide can be used both as therapeutics
and diagnostics. Antibodies are produced by immunologically challenging a
B-cell-containing biological system, e.g., an animal such as a mouse,
with a MANF polypeptide to stimulate production of anti-MANF by the
B-cells, followed by isolation of the antibody from the biological
system. Such antibodies can be used to measure MANF polypeptide in a
biological sample such as serum, by contacting the sample with the
antibody and then measuring immune complexes as a measure of the MANF
polypeptide in the sample. Antibodies to MANF can also be used as
therapeutics for the modulation of MANF biological activity.

[0024] Thus, in another aspect, the invention features a purified antibody
that specifically binds to a MANF polypeptide.

[0025] In yet another aspect, the invention features a method for
determining whether a candidate compound modulates MANF-mediated
dopaminergic neuronal survival-promoting activity, including: (a)
providing a MANF polypeptide; (b) contacting the MANF polypeptide with
the candidate compound; and (c) measuring MANF biological activity,
wherein altered MANF biological activity, relative to that of a MANF
polypeptide not contacted with the compound, indicates that the candidate
compound modulates MANF biological activity. The MANF polypeptide can be
in a cell or in a cell-free assay system.

[0026] In another aspect, the invention features a method for determining
whether candidate compound is useful for decreasing neurodegeneration,
the method including the steps of: (a) providing a MANF polypeptide; (b)
contacting the polypeptide with the candidate compound; and (c) measuring
binding of the MANF polypeptide, wherein binding of the MANF polypeptide
indicates that the candidate compound is useful for decreasing
neurodegeneration.

[0027] In particular embodiments of the foregoing screening methods of the
present invention, the cell is in an animal and the MANF polypeptide
consists of the sequence of SEQ ID NO: 2, 3, 4, 5, 6, or 7, or consists
essentially of SEQ ID NO: 2, 3, 4, 5, 6, or 7.

[0028] The invention also features screening methods for identifying
factors that potentiate or mimic MANF biological activity. In these
screening methods for potentiators, the ability of candidate compounds to
increase MANF expression, stability, or biological activity is tested
using standard techniques. A candidate compound that binds to MANF may
act as a potentiating agent. A mimetic (e.g., a compound that binds a
MANF receptor) is a compound capable of acting in the absence of a MANF
polypeptide.

[0029] By "substantially purified" is meant that a polypeptide (e.g., a
MANF polypeptide) has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially purified when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is at least 75%, more preferably
at least 90%, and most preferably at least 99%, by weight, pure. A
substantially purified polypeptide may be obtained, for example, by
extraction from a natural source (e.g., a neural cell), by expression of
a recombinant nucleic acid encoding the polypeptide, or by chemically
synthesizing the protein. Purity can be measured by any appropriate
method, e.g., by column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.

[0030] A polypeptide is substantially free of naturally associated
components when it is separated from those contaminants that accompany it
in its natural state. Thus, a polypeptide which is chemically synthesized
or produced in a cellular system different from the cell from which it
naturally originates will be substantially free from its naturally
associated components. Accordingly, substantially purified polypeptides
include those which naturally occur in eukaryotic organisms but are
synthesized in E. Coli or other prokaryotes.

[0031] By "polypeptide" or "protein" is meant any chain of more than two
amino acids, regardless of post-translational modification such as
glycosylation or phosphorylation.

[0033] A polynucleotide that is a part of the invention is one encoding a
MANF polypeptide, as defined above. Exemplary polynucleotides are
represented, for example, by the sequences of SEQ ID NO: 9 and SEQ ID NO:
10.

[0034] By "substantially identical" is meant a polypeptide or
polynucleotide exhibiting at least 5%, preferably 90%, more preferably
95%, and most preferably 97% identity to a reference amino acid or
nucleic acid sequence. For polypeptides, the length of comparison
sequences will generally be at least 16 amino acids, preferably at least
20 amino acids, more preferably at least 25 amino acids, and most
preferably 35 amino acids. For polynucleotides, the length of comparison
sequences will generally be at least 50 nucleotides, preferably at least
60 nucleotides, more preferably at least 75 nucleotides, and most
preferably 110 nucleotides.

[0036] By "high stringency conditions" is meant hybridization in
2×SSC at 40° C. with a DNA probe length of at least 40
nucleotides. For other definitions of high stringency conditions, see F.
Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6,
John Wiley & Sons, New York, N.Y., 1994, hereby incorporated by
reference.

[0037] By "compound" or "factor" is meant a molecule having an activity
that promotes the survival (or, conversely, prevents the death) of
dopaminergic neurons in a standard cell survival assay.

[0038] By "composition" is meant a collection of polypeptides, including a
polypeptide of the present invention.

[0039] By "pharmaceutically acceptable excipient" is meant an excipient,
carrier, or diluent that is physiologically acceptable to the treated
mammal while retaining the therapeutic properties of the polypeptide with
which it is administered. One exemplary pharmaceutically acceptable
carrier is physiological saline solution. Other physiologically
acceptable carriers and their formulations are known to one skilled in
the art and described, for example, in Remington: The Science and
Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippencott
Williams & Wilkins.

[0040] By a compound having "dopaminergic neuronal survival-promoting
activity" is the presence of the compound increases survival of
dopaminergic neurons by at least two-fold in a dopaminergic neuronal
survival assay (such as the one described herein) relative to survival of
dopaminergic neurons in the absence of the compound. The increase in the
survival of dopaminergic neurons can be by at least three-fold, more
preferably by at least four-fold, and most preferably by at least
five-fold. The assay can be an in vitro assay or an in vivo assay.

[0041] The present invention provides new methods and reagents for the
prevention of neuronal cell death. The invention also provides
pharmaceutical compositions for the treatment of neurological diseases or
disorders of which aberrant neuronal cell death is one of the causes.

[0042] Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 is a schematic illustration showing the effect of different
amounts of conditioned medium from VMCL-1 cell cultures on the survival
of tyrosine hydroxylase-positive cells (top panel) and MAP2-positive
cells (bottom panel).

[0054] We previously discovered that a cell line of mesencephalic origin
(termed "VMCL-1") secretes a factor that, in turn, promotes
differentiation and survival of dopaminergic neurons. This cell line
grows robustly in a serum-free medium. Moreover, the CM prepared from
these cells contains one or more dopaminergic neuronal survival factors
that increase the survival of mesencephalic dopaminergic neurons at least
3-fold, and promotes their development as well.

[0055] We purified, from the VMCL-1 cell line, a protein that we
identified to be ARP. We purified this protein as follows. A 3 L volume
of VMCL-1 conditioned medium was prepared, and subjected to five
sequential steps of column chromatography. At each purification step,
each column fraction was tested for biological activity in the bioassay
referred to above. An estimate of the effect of each fraction on
dopaminergic neuronal survival was done at 24 hour intervals, over a
period of five days, and rated on a scale of 1-10. After the fifth
purification step, the biologically active fraction and an adjacent
inactive fraction were analyzed by SDS-PAGE. The results of the SDS-PAGE
analysis revealed a distinctive protein band in the 20 kDa range in the
lane from the active fraction. The "active" band was excised and
subjected to tryptic digest, and the molecular mass and sequence of each
peptide above background were determined by mass spectrometry analysis.
The following two peptide sequences were identified: DVTFSPATIE (SEQ ID
NO: 6) and QIDLSTVDL (SEQ ID NO: 7). A search of the database identified
a match for human arginine-rich protein and its mouse orthologue. The
predicted protein encoded by the mouse EST sequence is about 95%
identical to the predicted human protein. A search of the rat EST
database revealed two sequences, one (dbEST Id: 4408547; EST name:
EST348489) having significant homology at the amino acid level to the
human and mouse proteins. The full-length rat sequence was not in the
GenBank database. Additionally, we discovered that the sequence of the
human ARP in GenBank was incorrect. The correct sequence is depicted in
SEQ ID NO: 1.

[0056] We have discovered that human ARP is cleaved such that the
arginine-rich amino-terminus is separated from the carboxy-terminus to
produce human pro-MANF (SEQ ID NO: 2). The cleaved carboxy-terminal
fragment contains a signal peptide, resulting in the secretion of human
MANF (SEQ ID NO: 3) from the cell.

[0057] Both the secreted form and the unsecreted form of MANF
(collectively referred to as MANF polypeptides) have neurotrophic
activity and are useful as neurotrophic factor for the treatment of a
neurodegenerative disease such as Parkinson's Disease and for improving
dopaminergic neuronal survival during or following transplantation into a
human. MANF polypeptides can also be used to improve the in vitro
production of neurons for transplantation. In another use, MANF
polypeptides can be used for the identification of compounds that
modulate or mimic MANF's dopaminergic neuronal survival-promoting
activity. MANF polypeptides can also be used to identify MANF receptors.
Each of these uses is described in greater detail below.

Identification of Molecules that Modulate MANF Biological Activity

[0058] The effect of candidate molecules on MANF-mediated regulation of
dopaminergic neuronal survival may be measured at the level of
translation by using standard protein detection techniques, such as
western blotting or immunoprecipitation with a MANF-specific antibody.

[0059] Compounds that modulate the level of MANF may be purified, or
substantially purified, or may be one component of a mixture of compounds
such as an extract or supernatant obtained from cells (Ausubel et al.,
supra). In an assay of a mixture of compounds, MANF expression is
measured in cells administered progressively smaller subsets of the
compound pool (e.g., produced by standard purification techniques such as
HPLC or FPLC) until a single compound or minimal number of effective
compounds is demonstrated to MANF expression.

[0060] Compounds may also be directly screened for their ability to
modulate MANF-mediated dopaminergic neuronal survival. In this approach,
the amount of dopaminergic neuronal survival in the presence of a
candidate compound is compared to the amount of dopaminergic neuronal
survival in its absence, under equivalent conditions. Again, the screen
may begin with a pool of candidate compounds, from which one or more
useful modulator compounds are isolated in a step-wise fashion.
Survival-promoting activity may be measured by any standard assay.

[0061] Another method for detecting compounds that modulate the activity
of MANF is to screen for compounds that interact physically with MANF.
These compounds may be detected by adapting interaction trap expression
systems known in the art. These systems detect protein interactions using
a transcriptional activation assay and are generally described by Gyuris
et al. (Cell 75:791-803, 1993) and Field et al., (Nature 340:245-246,
1989). Alternatively, MANF or a biologically active fragment thereof can
be labeled with 125I Bolton-Hunter reagent (Bolton et al. Biochem.
J. 133: 529, 1973). Candidate molecules previously arrayed in the wells
of a multi-well plate are incubated with the labeled MANF, then washed;
any wells with labeled MANF complex are assayed. Data obtained using
different concentrations of MANF can be used to calculate values for the
number, affinity, and association of MANF with the candidate molecules.

[0062] Compounds or molecules that function as modulators of MANF
dopaminergic neuronal survival-promoting activity may include peptide and
non-peptide molecules such as those present in cell extracts, mammalian
serum, or growth medium in which mammalian cells have been cultured.

[0063] A molecule that modulates MANF expression or MANF-mediated
biological activity such that there is an increase in neuronal cell
survival is considered useful in the invention; such a molecule may be
used, for example, as a therapeutic agent, as described below.

[0064] The discovery of MANF as a neurotrophic factor that promotes the
survival of dopaminergic neurons allows for its use for the therapeutic
treatment of neurodegenerative diseases such as Parkinson's disease.

[0065] To add a MANF polypeptide to cells in order to prevent neuronal
death, it is preferable to obtain sufficient amounts of a recombinant
MANF polypeptide from cultured cell systems that can express the protein.
A preferred MANF polypeptide is human MANF, but MANF polypeptides derived
from other animals (e.g., pig, rat, mouse, dog, baboon, cow, and the
like) can also be used. Delivery of the protein to the affected tissue
can then be accomplished using appropriate packaging or administrating
systems. Alternatively, small molecule analogs may be used and
administered to act as MANF agonists and in this manner produce a desired
physiological effect.

[0066] Gene therapy is another potential therapeutic approach in which
normal copies of the gene encoding a MANF polypeptide (or a
polynucleotide encoding MANF sense RNA) is introduced into cells to
successfully produce the MANF polypeptide. The gene must be delivered to
those cells in a form in which it can be taken up and encode for
sufficient protein to provide effective dopaminergic neuronal
survival-promoting activity.

[0068] Gene transfer could also be achieved using non-viral means
requiring infection in vitro. This would include calcium phosphate, DEAE
dextran, electroporation, and protoplast fusion. Liposomes may also be
potentially beneficial for delivery of DNA into a cell. Although these
methods are available, many of these are of lower efficiency.

[0069] In the constructs described, MANF or pro-MANF cDNA expression can
be directed from any suitable promoter (e.g., the human cytomegalovirus
(CMV), simian virus 40 (SV40), or metallothionein promoters), and
regulated by any appropriate mammalian regulatory element. For example,
if desired, enhancers known to preferentially direct gene expression in
neural cells may be used to direct MANF polypeptide expression. The
enhancers used could include, without limitation, those that are
characterized as tissue- or cell-specific in their expression.

[0070] RNA molecules may be modified to increase intracellular stability
and half-life. Possible modifications include, but are not limited to,
the addition of flanking sequences at the 5' and/or 3' ends of the
molecule or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the backbone of the molecule. This
concept can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as well
as acetyl-, methyl-, thio-, and similarly modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily
recognized by endogenous endonucleases.

[0071] Another therapeutic approach within the invention involves
administration of a recombinant MANF polypeptide, either directly to the
site of a potential or actual cell loss (for example, by injection) or
systemically (for example, by any conventional recombinant protein
administration technique).

[0072] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with a
pharmaceutically acceptable carrier, for any of the therapeutic effects
discussed above. Such pharmaceutical compositions may consist of AMNF
polypeptides, antibodies to MANF polypeptides, and/or mimetics and
agonists of MANF polypeptides. The compositions may be administered alone
or in combination with at least one other agent, such as stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier, including, but not limited to, saline, buffered
saline, dextrose, and water. The compositions may be administered to a
patient alone, or in combination with other agents, drugs or hormones.

[0073] In one example, a MANF polypeptide is administered to a subject at
the site that cells are transplanted. The administration of the MANF
polypeptide can be performed before, during, or after the transplantation
of the cells. Preferably, the two steps are within about four hours of
each other. If desirable, the MANF polypeptide can be repeatedly
administered to the subject at various intervals before and/or after cell
transplantation. This protective administration of the MANF polypeptide
may occur months or even years after the cell transplantation.

[0074] In addition to its administration to a human or other mammal, a
MANF polypeptide can also be used in culture to improve the survival of
neurons during their production any time prior to transplantation. In one
example, the cells to be transplanted are suspended in a pharmaceutical
carrier that also includes a survival-promoting amount of a MANF
polypeptide. A MANF polypeptide can also be administered to the cultures
earlier in the process (e.g., as the neurons are first differentiating).
It is understood that the neurons need not be primary dopaminergic
neurons. Neurons (e.g., dopaminergic neurons) that are differentiated,
either in vitro or in vivo, from stem cells or any other cell capable of
producing neurons can be cultured in the presence of a MANF polypeptide
during their production and maintenance.

[0075] Parenteral formulations may be in the form of liquid solutions or
suspensions; for oral administration, formulations may be in the form of
tablets or capsules; and for intranasal formulations, in the form of
powders, nasal drops, or aerosols.

[0076] Methods well known in the art for making formulations are to be
found in, for example, Remington: The Science and Practice of Pharmacy,
(20th ed.) ed. A. R. Gennaro Ark., 2000, Lippencott Williams & Wilkins.
Formulations for parenteral administration may, for example, contain as
excipients sterile water or saline, polyalkylene glycols such as
polyethylene glycol, oils of vegetable origin, or hydrogenated
naphthalenes, biocompatible, biodegradable lactide polymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control the
release of the present factors. Other potentially useful parenteral
delivery systems for the factors include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain as excipients, for example,
lactose, or may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be
oily solutions for administration in the form of nasal drops, or as a gel
to be applied intranasally.

[0077] The present factors can be used as the sole active agents, or can
be used in combination with other active ingredients, e.g., other growth
factors which could facilitate dopaminergic neuronal survival in
neurological diseases, or peptidase or protease inhibitors.

[0078] The concentration of the present factors in the formulations of the
invention will vary depending upon a number of issues, including the
dosage to be administered, and the route of administration.

[0079] In general terms, the factors of this invention may be provided in
an aqueous physiological buffer solution containing about 0.1 to 10% w/v
polypeptide for parenteral administration. General dose ranges are from
about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose
range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The
preferred dosage to be administered is likely to depend upon the type and
extent of progression of the pathophysiological condition being
addressed, the overall health of the patient, the make up of the
formulation, and the route of administration.

[0080] While human MANF is preferred for use in the methods described
herein, MANF has been identified in numerous species, including rat,
mouse, and cow. One in the art will recognize that the identification of
MANF from other animals can be readily performed using standard methods.
Any protein having dopaminergic neuronal survival-promoting activity and
encoded by a nucleic acid that hybridizes to the cDNA encoding human ARP
is considered part of the invention.

[0081] The following examples are to illustrate the invention. They are
not meant to limit the invention in any way.

Example 1

Production and Analysis of VMCL-1 Cells

[0082] The VMCL-1 cell line was made as follows. Rat E14 mesencephalic
cells, approximately 2-3% of which are glioblasts, were incubated in
medium containing 10% (v/v) fetal bovine serum for 12 hours and
subsequently expanded in a serum-free medium, containing basic fibroblast
growth factor (bFGF) as a mitogen. After more than 15 DIV, several islets
of proliferating, glial-like cells were observed. Following isolation and
passaging, the cells (referred to herein as VMCL-1 cells) proliferated
rapidly in either a serum-free or serum-containing growth medium.
Subsequent immunocytochemical analysis showed that they stained positive
for two astrocytic markers, GFAP and vimentin, and negative for markers
of oligodendroglial or neuronal lineages, including A2B5, O4, GalC and
MAP2. We have deposited the VMCL-1 cell line with the ATCC (Accession No:
PTA-2479; deposit date: Sep. 18, 2000).

[0083] Serum-free CM, prepared from the VMCL-1 cells, caused increased
survival and differentiation of E14 mesencephalic dopaminergic neurons in
culture. These actions are similar to those exerted by CM derived from
primary, mesencephalic type-1 astrocytes. The expression of mesencephalic
region-specific genes (e.g., wnt-1, en-1, en-2, pax-2, pax-5 and pax-8),
was similar between VMCL-1 cells and primary, type-1 astrocytes of E14
ventral mesencephalic origin. In both, wnt-1 was expressed strongly, and
en-1 less strongly, supporting an expression pattern expected of their
mesencephalic origin. A chromosomal analysis showed that 70% of the cells
were heteroploid, and of these, 50% were tetraploid. No apparent decline
in proliferative capacity has been observed after more than twenty-five
passages. The properties of this cell line are consistent with those of
an immortalized, type-1 astrocyte.

[0084] The VMCL-1 cells have a distinctly non-neuronal, glial-like
morphology, but lack the large, flattened shape that is typical of type-1
astrocytes in culture Immunocytochemical analysis demonstrated that they
stained positive for GFAP and vimentin, and negative for MAP2, A2B5 and
O4. The cells were therefore not of the oligodendrocyte lineage. On the
basis of a negative reaction to A2B5 and their morphological
characteristics they were also not type-2 astrocytes. The classification
that is supported by the immunocytochemical evidence is of type-1
astrocytes, although, as noted, these cells lack the classical
morphological traits of primary type-1 astrocytes in culture.

Example 2

Action of VMCL-1 CM on E14 Dopaminergic Neurons in Culture

[0085] VMCL-1 CM was tested at 0, 5, 20 and 50% v/v, for its ability to
influence survival and development of E14 mesencephalic dopaminergic
neurons in culture. The cultures were primed with 10% fetal bovine serum
(FBS) for 12 hours, then grown in a serum-free growth medium thereafter,
until they were stained and analyzed after 7 DIV. There was a
dose-dependent action of the CM on the increased survival of dopaminergic
neurons. The CM increased survival by 5-fold. In contrast, there was no
significant increase in non-dopaminergic neuronal survival. The profile
of the biological action of this putative factor is quite different from
that of CM derived from the B49 glioma cell line, the source of GDNF (Lin
et al., Science 260: 1130-1132).

Example 3

Gene Expression Analysis of VMCL-1 Cells

[0086] To further investigate the similarity between the VMCL-1 cell line
and primary cultured astrocytes, we measured the expression of six marker
genes characteristic of the mesencephalic region. Analysis of wnt-1,
en-1, en-2, pax-2, pax-5, and pax-8 showed that all genes were expressed
in both E13 and E14 ventral mesencephalon neural tissue, with the
exception of pax-2, which was expressed at E13 but not E14 neural tissue.
Both primary astrocytes and VMCL-1 cells expressed wnt-1 at levels
comparable with those of E13 and E14 ventral mesencephalic neural tissue.
The degree of expression of en-1 was similar in primary astrocytes and
VMCL-1 cells, although at a lower level versus expression in E13 and E14
ventral mesencephalic tissue. In contrast, en-2, pax-5 and pax-8 were not
expressed in either primary astrocytes or VMCL-1. Pax-2 was expressed in
E13 but not E14 ventral mesencephalon, and in primary astrocytes, but not
in VMCL-1.

Example 4

Chromosomal Analysis of VMCL-1 Cells

[0087] Chromosomes were counted in 34 cells. Of these, 9 had a count of
42, the diploid number for rat. Of the 25 cells that were heteroploid,
12/25 or 48% were in the tetraploid range. Hyperdiploid (counts of 43-48)
and hypodiploid (counts of 39-41) cells each accounted for 20% of the
population, while 12% of the cells had structurally rearranged
chromosomes.

[0088] The selective action of VMCL-1 CM in increasing the survival of
dopaminergic neurons in culture provides a potential clinical use for the
molecule(s) produced by this cell line. The lack of a toxic action of
VMCL-1 CM at a concentration of 50% v/v indicates that the active,
putative neurotrophic factor is not toxic. The action exerted by VMCL-1
CM mirrors almost exactly that of CM prepared from mesencephalic, primary
type-1 astrocytes (Takeshima et al., J. Neurosci. 14: 4769-4779, 1994). A
high degree of specificity of the putative factor from VMCL-1 for
dopaminergic neurons is strongly indicated from the observation that
general neuronal survival was not significantly increased, while the
survival of dopaminergic neurons was increased 5-fold (FIG. 1). We have
demonstrated that primary type-1 astrocytes express GDNF mRNA, but have
not detected GDNF protein by Western blot in the CM, at a sensitivity of
50 pg. Moreover, we have shown that under the present experimental
conditions, the increased survival of dopaminergic neurons mediated by an
optimal concentration of GDNF is never greater than 2-fold. These
observations alone indicate that the factor responsible for the
neurotrophic actions of VMCL-1 CM is not GDNF.

Example 5

Production of Type-1 Astrocyte-Conditioned Medium

[0089] E16 type-1 astrocyte CM (10 L) was filtered and applied to a
heparin sepharose CL-6B column (bed volume 80 mL) which had previously
been equilibrated with 20 mM Tris-HCl (Mallinckrodt Chemical Co. Paris,
Ky.) pH 7.6 containing 0.2 M NaCl. After washing with equilibration
buffer, bound proteins were eluted from the column with a linear gradient
of 0.2 M-2 M NaCl in 20 mM Tris-HCl pH 7.6 (400 mL total volume, flow
rate 100 mL/hr). Fractions were collected using a Pharmacia LKB fraction
collector and absorbance was measured at 280 nm (Sargent-Welch PU 8600
UV/VIS Spectrophotometer). A 1 mL aliquot was taken from each fraction,
pooled into groups of four (4 mL total volume) and desalted using
Centricon-10® membrane concentrators (Millipore, Bedford, Mass.).
Samples were diluted 1:4 in defined medium and bioassayed for
dopaminergic activity. Active fractions were pooled (80 mL total volume)
and then applied to a G-75 Sephadex® column (70×2.5 cm,
Pharmacia Biotechnology Ltd., Cambridge, UK) which had been
pre-equilibrated with 50 mM ammonium formate pH 7.4. Proteins were
separated with the same buffer (flow rate, 75 mL/hr) and absorbance was
measured at 280 nm A 1 mL aliquot was taken from each fraction, pooled
into groups of four (4 mL total volume), concentrated by lyopholyzation
and reconstituted in 1 mL distilled water volume. Samples were then
diluted 1:4 in defined medium for dopaminergic bioassay. Those with
neurotrophic activity were further bioassayed as individual fractions.

[0090] An important distinguishing feature of VMCL-1 CM is that it
promotes predominantly the survival of dopaminergic neurons, compared
with the survival of GABAergic, serotonergic, and other neuronal
phenotypes present in the culture. This claim of specificity is also made
for GDNF. The results of extensive testing have demonstrated, however,
that the VMCL-1-derived compound is not GDNF.

Example 6

Isolation and Purification of a Protein Having Dopaminergic Neuronal
Survival-Promoting Activity

[0091] The purification protocol was performed as follows. All salts used
were of the highest purity and obtained from Sigma Chemical Co. All
buffers were freshly prepared and filtered via 0.2 μM filter (GP
Express vacuum-driven system from Millipore)

[0094] The active fractions from step 2 that corresponded to the 20-30 kDa
elution region were pooled and concentrated to 7.5 mL, using a Centricon
Plus-20 concentrator (5,000 MWCO), dialyzed overnight at 4° C.
against 2 L of 10 mM sodium phosphate buffer, pH 7.2 (buffer A) and
loaded (via Superloop) onto a 1 mL pre-packed ceramic hydroxyapatite
(Type I, Bio-Rad) column equilibrated with buffer A. After the excess of
unbound protein (flow through) was washed off the column with buffer A,
the linear gradient of buffer A containing 1.0 M NaCl was applied from 0
to 100%. One milliliter fractions were collected and analyzed for
activity. The active protein was eluted as a broad peak within the region
of gradient corresponding to 0.4-0.8 M NaCl concentration.

[0096] The active protein fraction from Step 4 (7 mL of total volume) was
concentrated down to nearly zero volume (about 1 μL) using Centricon
Plus-20 concentrator (5,000 MWCO) and reconstituted in 0.6 mL of 10 mM
sodium phosphate buffer, pH 7.2. The reconstituted material (70 μL,
analytical run) was loaded onto BioSil 125 HPLC gel-filtration column
(Bio-Rad) equilibrated with 20 mM sodium phosphate buffer, pH 7.2 (GF
buffer). The chromatography was conducted using HP 1100 Series HPLC
system (Hewlett-Packard). The eluate was collected in 120 μL fractions
and analyzed for activity and protein content (SDS-PAGE). The activity
was found in fractions associated with the main 280-nm absorbance peak
eluted from the column, which was represented by a 45-kDa protein
according to SDS-PAGE analysis. Nevertheless, no activity was found in
the side fractions of the 45-kDa protein peak, indicating that activity
might be due to the presence of another protein that was co-eluted with
45 kDa protein, but at much lower concentration that could not be
detected on the 12% SDS-PAGE silver-stained gel. Therefore, the remaining
concentrated material from step 5 was further concentrated down to 80
μL volume using a Centricon-3 concentrator (Millipore), and 60 μL
was loaded and separated on the column at the same conditions as for the
above-described analytical run. Aliquots of 8 μL were taken from each
120 μL fraction of the eluate and analyzed by SDS-PAGE (12% gel)
combined with silver staining. This analysis indicated that another two
additional proteins (having molecular weights of about 18 and 20 kDa)
were associated with the active fractions and co-eluted with the major
45-kDa protein. The active fractions were dialyzed against 1 L of
ammonium acetate buffer, pH 8.0 (4° C.) and combined to create two
active pools, P-1 and P-2, such that P-1 contained the 20 kDa protein and
the 45 kDa protein, and P-2 contained the 18 kDa protein and the 45 kDa
protein. Each pool was dried down on SpeedVac vacuum concentrator
(Savant) and separately reconstituted in 15 μL 0.1 M ammonium acetate
buffer, pH 6.9. Aliquots were withdrawn from each sample and assayed for
activity. Additionally, 1 μL aliquots were subjected to 12% SDS-PAGE
analysis followed by silver staining.

[0097] The results of the foregoing analysis clearly indicated that P-1,
but not P-2, contained the desired survival-promoting activity. In the
next step, both P-1 and P-2 were dried on SpeedVac, reconstituted (each)
in 10 μL of freshly prepared SDS-PAGE reducing sample buffer
(Bio-Rad), incubated for one minute in a boiling water bath and loaded
onto a 12% SDS-PAGE gel. After electrophoresis was complete, the gel was
fixed in methanol/acetic acid/water solution (50:10:40) for 40 minutes at
room temperature, washed three times with nanopure water, and stained
overnight with GelCode Blue Stain Reagent (Pierce) at room temperature.
After staining was completed, and the GelCode solution was washed off the
gel with nanopure water, the visible protein bands corresponding to the
45 kDa protein (both P-1 and P-2) and the 20 kDa protein (P-1 only) were
excised from the gel with a razor blade. Each gel slice containing a
corresponding band was placed in a 1.5 mL microcentrifuge tube until the
time of in-gel digestion.

Example 7

Analysis of In-Gel Digested Fragments by nESI-MS/MS

[0098] The protein gel bands were incubated with 100 mM ammonium
bicarbonate in 30% acetonitrile (aq.) at room temperature for 1 hour in
order to remove the colloidal comassie blue stain. The destaining
solution was replaced a number of times until the dye was completely
removed. The gel pieces were then covered with deionized water
(˜200 μL) and shaken for 10 minutes. The gel pieces were
dehydrated in acetonitrile and, after removing the excess liquid, were
dried completely on a centrifugal evaporator. The gel bands were
rehydrated with 20 μL of 50 mM ammonium bicarbonate, pH 8.3,
containing 200 ng of modified trypsin (Promega, Madison, Wis.). The gel
pieces were covered with 50 mM ammonium bicarbonate, pH 8.3
(approximately 50 μL), and were incubated overnight at 37° C.
The digest solutions were then transferred to clean eppendorf tubes and
the gel pieces were sonicated for 30 minutes in 50-100 μL of 5% acetic
acid (aq). The extract solutions were combined with the digest solutions
and evaporated to dryness on a centrifugal evaporator.

[0099] The in-gel digest extracts were first analyzed by matrix-assisted
laser desorption ionization-time of flight mass spectrometry
(MALDI-TOFMS) using a Voyager Elite STR MALDI-TOFMS instrument (Applied
Biosystems Inc., Framingham, Mass.). The extracts were dissolved in 5
μL of 50% acetonitrile, 1% acetic acid. Dihydroxybenzoic acid was used
as the matrix and spectra were acquired in positive ion, reflectron mode.
Approximately one fifth of each sample was used for this analysis. These
spectra provided the masses of the peptides in the digest extracts which
were then used to search an in-house, non-redundant protein sequence
database, a process called peptide mass fingerprinting. The remainder of
the samples were used for peptide sequencing analysis by nanoelectrospray
ionization-tandem mass spectrometry (nESI-MS/MS). The extracts were first
desalted using C18 ZipTips (Millipore) and redissolved in 75% methanol
(aq.), 0.1% acetic acid (5 μL). Approximately one half of the samples
were loaded into nanoelectrospray glass capillaries (Micromass).
nESI-MS/MS analyses were carried out using a Q-Star quadrupole
time-of-flight hybrid mass spectrometer (PE SCIEX, Concord, ON). All
MS/MS analyses were carried out in positive ion mode. The collision gas
was nitrogen and the collision energy was 40-60 eV. MS/MS spectra were
typically acquired every second over a period of two minutes. The MS/MS
spectra were used to search an in-house non-redundant protein sequence
database using partial sequence tags (i.e., only the peptide mass and a
few fragment ions are used to search the database). If the protein was
not identified by this procedure then the amino acid sequences of two or
more peptides were determined as fully as possible from the MS/MS
spectra. These sequences were used to carry out BLAST searches on NCBI's
protein, nucleotide and EST sequence databases.

Example 8

Identification of MANF, a Secreted Form of ARP

[0100] In order for ARP to be a factor that is responsible at least in
part for the observed neurotrophic activity of VMCL-1 CM, the protein
must be released from the cell. The predicted amino terminus of ARP has
basic charges, however, a property that would favor retention in the cell
nucleus. Nonetheless, we hypothesized that there would also exist a
secreted form.

[0101] Support for our hypothesis was found in a publication by Goo et al.
(Molecules and Cells 9:564-568, 1999), who identified a cDNA encoding an
ARP-like protein in Drosophila melanogaster while screening a cDNA
library using a yeast signal sequence trap technique. The putative
ARP-like protein encoded by this cDNA lacks the arginine-rich amino
terminus. Using the SignalP program, we identified a signal peptide
(residues 1-22) and a signal peptidase-cutting site between alanine 22
and leucine 23 of Drosophila ARP-like protein, providing additional
evidence that Drosophila ARP-like protein is secreted.

[0102] Based on the alignment between human ARP and Drosophila ARP-like
protein, we postulated that human ARP would have a signal sequence and
signal peptidase cutting site. Accordingly, we used the SignalP program
to analyze the human ARP lacking the arginine-rich amino terminus (amino
acids 1-55); this polypeptide is now referred to as pro-MANF. In this
example, the methionine at position 56 is the start codon. The SignalP
program predicted a signal peptide consisting of residues 1-21 of SEQ ID
NO: 2 and a cutting site between alanine 21 and leucine 22, which is
consistent with the results from the analysis of the Drosophila ARP-like
protein. The predicted cleaved human MANF protein is depicted in FIG. 2
and SEQ ID NO: 3. This and other exemplary MANF polypeptides are shown in
FIGS. 2 and 3. Exemplary MANF polynucleotides are shown in FIG. 4.

[0103] Based in part on our analysis of Drosophila ARP-like protein
(GenBank Accession No. AF132912--1) and human ARP, we predict that
the translation can begin at either the methionine at position 1 or the
methionine at position 56 of human ARP. In the latter case, the signal
peptide-containing protein (pro-MANF) is capable of being secreted from
the cell in the form of MANF, where the protein acts a neurotrophic
factor. Our discovery of the existence of MANF does not, however,
preclude an intracellular function for the ARP containing the
arginine-rich amino-terminal region.

Example 9

Biological Activity of MANF Expressed in E. Coli

[0104] Recombinant protein expression was carried out in E. Coli bacterial
cells using pTriEx containing a polynucleotide encoding human MANF (SEQ
ID NO: 3). A total of 4 mg of purified recombinant MANF was obtained from
350 mL of bacterial cell culture, its identity confirmed by mass spec
sequencing. This protein was tested for its ability to protect DA
neurons. As shown in FIG. 5, MANF expressed in E. Coli was capable of
protecting DA neurons from cell death to the same extent as did the
VMCL-1 conditioned medium.

Example 10

Dose-Response for Eukaryotic MANF Expressed in HEK293 Cells

[0105] The dose-relationship of human MANF (99% pure, produced in HEK293
cells) versus survival of dopaminergic neurons was tested using a
dopaminergic cell culture assay system containing 20% of dopaminergic
neurons. E14 pregnant rats were killed by CO2 narcosis. The torso
was soaked in 70% EtOH, a laporatomy was performed, and the uterine sac
removed and transferred to a 50 mL tube containing 20 mL cold HBSS, pH
7.4. Each uterine sac was in turn transferred to a 10-cm petri dish
containing 15 mL cold HBSS. The fetuses were removed intact, and each
brain was isolated intact and transferred to a new 10-cm petri dish
containing 15 mL cold HBSS. The medial ventral mesencephalon (VM) at the
roof of the mesencephalic flexure was dissected to obtain 1.0 mm3
piece of tissue at a packing density of 1.0×105
cells/mm3. The VM tissue was transferred to a 15 mL tube containing
10 mL of cold PCM10. The pooled VM tissue was washed with PCM10 (DMEM/F12
with 2 mM glutamine, 5 mg/mL insulin, 5 mg/mL transferrin, 5 mg/mL sodium
selenite; 20 nM progesterone, 30 nM thyroxine, and 10% fetal bovine
serum) (three washes), followed by a single wash in serum-free medium
(PCM0; same as PCM10 except that it lacks fetal bovine serum) and
digested in 2.0 ml of PCM0 containing papain (10 U/mL) for 15 minutes, at
37° C. The tissue was then rinsed (3×5 mL) with PCM10, to
inactivate the protease activity. Trituration was done in 2.0 mL of PCM0,
using a P-1000 set at 500 μL. The end point is a milky suspension with
no signs of tissue clumps. The dispersed cells were centrifuged (1,000
rpm, 2 min, 4° C.), counted, then resuspended at a density of
6.25×105 cells/mL in PCM10. Cell viability was tested at this
stage, and was usually >95%.

[0106] The cells were plated as microisland (MI) droplets of 25 μL,
(1.56×104 cells/MI) on 8-well chamber slides, coated with
poly-D-lysine. A 25 μL MI droplet occupies an area of 12.5 mm2.
The average, final, mean cell density of the MI is therefore
1.25×105 cells/cm2. The mean cell density at the center
of the MI is about 2.0×105/cm2, falling off to
<1.0×104 at the periphery of the MI. The MIs were incubated
at 37° C., in 5% CO2 at 100% humidity for 45 minutes to allow
the cells to attach to the coated surface. After attachment, 375 μL of
PCM10 was added to each well, and the cells serum-primed for 4 hr. At the
end of priming, 100% of PCM10 was aspirated, and replaced with
serum-free, PCM0.

[0108] Cultures were treated on the first, third and fifth days with the
indicated amount of MANF. The cultures were fixed and stained on DIV6 or
DIV7, using either the Vector ABC method, or indirect immunofluorescence.

[0109] As early as DIV3, there was a significant difference between the
different concentrations of MANF tested (ANOVA, P<0.001) (FIG. 6).
Paired comparisons using the Tukey method of analysis, indicated that
MANF at 250 and 500 pg/ml and 1.0 and 10 ng/nL were significantly
different from controls (P<0.05).

Example 11

Rank Order of Potency among BDNF, GDNF and MANF

[0110] As illustrated in FIG. 7, when three equivalent doses of BDNF, GDNF
and MANF were tested, the rank order of potency was:
MANF>GDNF>BDNF, indicating that the two lower concentrations of
MANF were more selective for DA neurons, relative to low doses of BDNF or
GDNF. At the highest dose, the rank order of potency was:
GDNF>MANF>BDNF. In general, BDNF tended to be the most potent, but
least specific for protecting DA neurons. At lower concentrations, MANF
tended to be the most selective in protecting DA neurons.

Example 12

Domains of MANF Predicted to be Active

[0111] We have identified three peptides from MANF that we predict may
have MANF biological activity (i.e., protect DA neurons from cell death).
The human peptides are LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFCREA; SEQ
ID NO: 11; RGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEKIC E KLKKKDSQICEL; SEQ ID
NO: 12 and KYDKQIDLSTVDLKKLRVKELKKI LDDWGETCKGCAEKSDYIRKINELMPKY; SEQ ID
NO: 13. Counterpart peptides can be readily identified using standard
sequence alignment programs. MANF sequences for mouse, cow, and pig are
provided herein. These peptides can be employed in any of the therapeutic
methods described herein, and are expressly considered to be "MANF
polypeptides."

Example 13

Selectivity of Responsiveness to MANF

[0112] The ability of MANF to protect neurons from other brain regions was
tested. No protection of rat cerebellar granule neurons, nodose sensory
neurons, or sympathetic noradrenergic neurons was observed. Similarly, in
ventral mesencephalic cultures, there appeared to be no activity on
GABAergic and serotonergic neurons in cultures in which MANF was
demonstrably protective for DA neurons. In contrast to the foregoing
results, MANF was protective for a subset of dorsal root ganglion cells
in culture. Dorsal root ganglia consist of at least three sub-populations
of neurons. It has been demonstrated that NGF, BDNF and NT-3, all members
of the neurotrophin family of neurotrophic factors, each acts on a
different subset of these neurons. The action of MANF on this subset of
dorsal root ganglion neurons, is therefore in keeping with the general
neuroprotective properties of neurotrophic factors.

Example 14

Production of MANF Polyclonal Antibodies

[0113] Polyclonal antibodies were prepared as follows. His-tagged full
length MANF was prepared in E. Coli. Six antigen injections of 200 μg
of purified MANF protein per injection per rabbit were performed (one
each on days 1, 21, 35, 49, 63, and 70). The serum was collected on day
84 (100 mL serum/rabbit).

[0114] Western blot analysis was used to test the activity of MANF-pAb. A
relatively high quantity of MANF (720 ng) was used for the initial test
of the activity of MANF-pAb, which remained active at a dilution of
1:12,800 (FIG. 8; lane 9). In the next test, the dilution of MANF was
fixed at 5,000 and the quantity of MANF varied from 1,000 to 15.6 ng. The
lowest quantity of MANF, 15.6 ng, was easily detected (FIG. 9, lane 9).
In tests for cross reactivity with BDNF and GDNF, the results showed that
even at three times the quantity of MANF (32 ng), the MANF-pAb did not
cross react with either BDNF or GDNF (FIGS. 10A-10C).

[0115] The foregoing results were obtained with the following methods.

Mesencephalic Cultures

[0116] Primary mesencephalic cell culture was prepared from timed-pregnant
Sprague-Dawley rats (Taconic Farms; Germantown, N.Y.). as described
previously (Shimoda et al., Brain Res. 586:319-323, 1992; Takeshima et
al., J. Neurosci. 14:4769-4779, 1994; Takeshima et al., Neuroscience.
60:809-823, 1994; Takeshima et al., J. Neurosci. Meth. 67:27-41, 1996).
The dissected tissue was collected and pooled in oxygenated, cold
(4° C.), HBSS or medium containing 10% fetal bovine serum
(Biofluids Laboratories, Rockville, Md.), depending on the purpose of the
experiment. Pregnant rats were killed by exposure to CO2 on the
fourteenth gestational day (i.e., E14), the abdominal region was cleaned
with 70% EtOH, a laparotomy was performed, and the fetuses collected and
pooled in cold Dulbecco's phosphate-buffered saline (DPBS), pH 7.4,
without Ca2+ or Mg2+. The intact brain was then removed, a cut
was made between the diencephalon and mesencephalon, and the tectum slit
medially and spread out laterally. The ventral, medial 1.0 mm3 block
of tissue comprising the mesencephalic dopaminergic region was isolated.
Dissected tissue blocks were pooled in cold (4° C.), oxygenated
medium. The tissue was triturated without prior digestion. Alternatively,
the cells were incubated in L-15 growth medium containing papain (Sigma
Chemical Co.), 10 U/mL, at 37° C., for 15 minutes, washed
(3×2 mL) with medium, and triturated using only the needle and
syringe. The dispersed cells were transferred to 1.5 mL Eppendorf tubes
(1.0 mL/tube), and centrifuged at ˜600 g for 2 minutes. The use of
higher centrifugation speeds for longer periods results in contamination
of the cultures with debris and, as a result, suboptimal growth of the
cells. The medium was carefully aspirated, and the cells resuspended in
fresh medium and counted using a hemocytometer. All procedures, from
laparotomy to plating were completed within 2 hours. In a typical
experiment, one litter of 10-15 fetuses yielded 1.0×105
cells/fetus, or 1.0×106-1.5×106 cells/litter.

Mesencephalic Microisland Cultures

[0117] To make mesencephalic microisland cultures, cells were prepared as
described above, and resuspended at a final density of 5.0×105
mL. A 25 uL droplet of the suspension (1.25×104 cells) was
plated using a 100 μL pipette onto 8-well chamber slides coated with
poly-D-lysine (50 μg/mL). The area of the droplet was ˜12.5
mm2, for a final mean cell density of 1.0×105/cm2.
The droplet was dispensed uniformly, and the pipette tip withdrawn
vertically, to avoid smearing. The area occupied by the microisland
culture remained uniform for the duration of the culture. The cultures
were incubated for 30 minutes at 37° C., in 5% CO2 at 100%
humidity, to allow the cells to attach, and 375 μL of growth medium
was then added to each well. The medium was changed after the first 12
hours, and approximately half of the medium was changed every second day
thereafter.

Cell Viability Assay

[0118] A two-color fluorescence cell viability assay kit (Live/Dead
Viability/Cytotoxicity Assay Kits, #L-3224, Molecular Probes, Inc.,
Eugene, Oreg.) was used to identify live and dead cells prior to plating
and in cultures. Live and dead cells fluoresced green and red,
respectively, giving two positive indicators of viability. Ethidium
homodimer and calcein-AM were diluted with DPBS to give final
concentrations of 3.8 μM and 2.0 μM, respectively. Evaluation of
cell viability was done before plating. A cell suspension was incubated
for 15 minutes with an equal volume of dye (typically 20 μL) on glass
slides at room temperature in a dark, humid chamber, coverslipped, and
then examined with a fluorescent microscope using FITC optics. Cell
viability just before plating was about 95%.

[0120] In preparing conditioned medium from the VMCL-1 cell line,
2.0×106 cells were plated in a 15 cm uncoated culture dish, in
20 mL of growth medium containing 1.0% of FBS. At 80% confluence, the
medium was aspirated and the cells washed once with serum-free medium. 20
mL of serum-free N2 medium without albumin was added, and conditioning
allowed to continue for 48 hours. During this time, the cells usually
expanded to 100% confluence. The medium was aspirated, pooled in 50 mL
tubes, centrifuged (15,000 rpm for 20 minutes) and subsequently pooled in
a 1.0 L plastic bottle. Usually 5 mL of each batch of CM was
filter-sterilized using a 0.22 μm filter, stored at aliquots of 5 mL,
at -70° C., and used to determine neurotrophic potency, before
being pooled with the larger store of CM. If desired, VMCL-1 CM can be
made in large quantities using standard industrial cell culture
techniques known to those in the art.

Production of Conditioned Medium for Type-1 Astrocytes

[0121] Type-1 astrocytes were prepared as follows. E16 rat fetal brain
stem was dissected in cold DPBS, and the mesencephalic region transferred
to astrocyte culture medium (DMEM/Ham's F-12, 1:1, 15% FBS, 4.0 mM
glutamine, 30 nM sodium selenite, penicillin, and streptomycin). Cells
were dispersed by trituration in 2 mL of fresh medium using an 18-gauge
needle fitted to a syringe. Cells were centrifuged for 5 minutes at 2,000
rpm in a centrifuge, re-suspended in medium, and triturated again. The
final cell pellet was dispersed and plated at a density of
1×106 cells/75 cm2 flask in 15 mL of medium. Cells were
incubated at 37° C. in an atmosphere of 5% carbon dioxide and 95%
air for 24 hours, and unattached cells were removed by aspiration. Cells
were cultured for an additional nine days, and flasks were then shaken
vigorously for 16 hours to remove any contaminating cell types. Astrocyte
monolayers were washed three times with DPBS, trypsinized and replated
(density of 1×106 cells/flask). At this time, a small
proportion of the cells were plated onto eight-well chamber slides (Nunc
Inc., Naperville, Ill.); these sister cultures were treated as described
for the flask cultures. At confluence, the medium was replaced with
medium containing 7.5% FBS and cells were incubated for 48 hours. At the
next exchange, defined serum-free medium (DMEM/Ham's F-12, 1:1, 4.0 mM
glutamine, 30 nM sodium selenite, penicillin 100 U/ml and streptomycin
100 U/mL) was added and cells were incubated for a further 48 hours.
Medium was replaced and, after five days, conditioned medium was
harvested and mixed with leupeptin (10 mM: Bachem, Torrance, Calif.) and
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1.0 mM: ICN
Biochemicals, Aurora, Ohio) to inhibit proteolysis. At the time of
harvesting, astrocyte monolayers cultured on chamber slides were
immunostained in order to assess the culture phenotype.

Culturing of VMCL-1 Cells

[0122] In culturing VMCL-1 and preparing VMCL-1 CM, 2.0×106
cells were plated in a 15-cm uncoated culture dish, in 20 mL growth
medium initially containing 10% FBS. At 80% confluence, the medium was
aspirated and the cells washed once with serum-free medium. Usually 20 mL
of serum-free medium without albumin was added, and conditioning allowed
to continue for 48 hours. N2 medium proved to be particularly suitable
for use to collect conditioned medium. During these 48 hours, the cells
usually expanded to 100% confluence. The medium was aspirated, pooled in
50 mL tubes, centrifuged (15,000 rpm, 20 min) and pooled in a 1.0 L
plastic bottle. Approximately 5 mL of each batch of CM was sterilized
using a 0.22 mm filter, stored at aliquots of 0.5 mL, at -70° C.,
and used to determine neurotrophic potency, before being pooled with the
larger store of CM. The VMCL-1 cell line has now been passaged greater
than 50 times.

Immunocytochemistry

[0123] For MAP2 and TH immunocytochemistry, the cultures were washed
(2×250 μL) with cold DPBS, fixed with 4% formaldehyde in PBS for
10 minutes, permeabilized using 1% CH3COOH/95% EtOH at -20°
C., for 5 minutes, and then washed (3×250 μL) with PBS.
Non-specific binding was blocked with 1% bovine serum albumin in PBS
(BSA-PBS) for 15 minutes. Anti-TH antibody (50 μL)
(Boehringer-Mannheim, Indianapolis, Ind.), or anti-MAP2 antibody
(Boehringer-Mannheim) was applied to each well, and the slides incubated
in a dark humid box at room temperature for 2 hours. Control staining was
done using mouse serum at the same dilution as the anti-TH antibody.
After washing (2×250 μL) with PBS, anti-mouse IgG-FITC (50
μL) was applied, and the slides incubated for an additional 1 hour.
After washing with PBS (2×250 μL), excess fluid was aspirated,
the chamber walls removed, and a single drop of VectaShield mounting
medium (Vector Laboratories, Burlingame, Calif.) applied, followed by a
cover glass, which was sealed with nail polish. In some experiments, TH
was identified using biotinylated, secondary antibodies, and the
nickel-enhanced, diaminobenzidine (DAB) reaction product was developed
using the biotinylated peroxidase-avidin complex (ABC kit; Vector
Laboratories).

[0124] For glial fibrillary acidic protein (GFAP, Boehringer-Mannheim,
#814369), fixation and permeabilization were done in one step using 5%
CH3COOH/95% C2H5OH at -20° C. The subsequent
procedures were the same as those used to visualize TH. For A2B5 and O4,
the cultures were washed with cold DPBS (2×250 μL) and blocked
with 1% BSA-PBS for 10 minutes. The A2B5 antibody (50 μL) was applied
to each well, and incubated for 1 hour. After washing with DPBS
(2×250 μL), the secondary antibody, anti-IgM-FITC, was applied
for 30 minutes. The cells were then washed with DPBS (2×250 μL).
To counter-stain cell nuclei, cells were incubated with 0.5 μg/mL of
nucleic acid dye H33258 (Hoechst, Kansas City, Mo.) in 10 mM sodium
bicarbonate for 15 minutes at room temperature, then rinsed in PBS for
2×10 minutes. After a final washing with cold DPBS (2×250
μL), they were mounted as described above.

RT-PCR Analysis

[0125] Total RNA was extracted from rat E13 or E14 ventral mesencephalic
tissue or from 1×109 astrocytes or from 1×109
VMCL-1 cells using RNA-STAT reagent (TelTest, University of Maryland,
Baltimore, Md.). First strand cDNA was generated from RNA and amplified
by polymerase chain reaction using the manufacturer's procedures.

[0126] Reaction products were resolved by 2% agarose gel electrophoresis
to determine size and relative abundance of fragments. PCR results for
b-actin and GAPDH were included as controls to confirm equal loading of
cDNA.

Chromosomal Analysis

[0127] The cells were grown in DMEM/F-12 1:1 medium supplemented with 2.5%
FBS, D-glucose (2.5 g/L) and ITS supplement, diluted 1:100. Twenty-four
hours later, subcultures at metaphase stage were arrested with colchicine
(10 μg/mL). The cells were trypsinized and subjected to hypotonic
shock (75 mM KCl). The cells were then fixed in three changes of
MeOH/CH3COOH, 3:1, and air-dried. The cells were then stained using
4% Geisma, and microscopically examined

Deposit

[0128] Applicant has made a deposit of at least 25 vials containing cell
line VMCL-1 with the American Type Culture Collection, Manassas Va.,
20110 U.S.A., ATCC Deposit No. PTA-2479. The cells were deposited with
the ATCC on Sep. 18, 2000. This deposit of VMCL-1 will be maintained in
the ATCC depository, which is a public depository, for a period of 30
years, or 5 years after the most recent request, or for the effective
life of the patent, whichever is longer, and will be replaced if it
becomes nonviable during that period. Additionally, Applicant has
satisfied all the requirements of 37 C.F.R. §§1.801-1.809,
including providing an indication of the viability of the sample.
Applicant imposes no restrictions on the availability of the deposited
material from the ATCC. Applicant has no authority, however, to waive any
restrictions imposed by law on the transfer of biological material or its
transportation in commerce. Applicant does not waive any infringement of
its rights granted under this patent.

Other Embodiments

[0129] All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will be
apparent to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be understood
that the invention should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes for
carrying out the invention which are obvious to those skilled in
molecular biology or related fields are intended to be within the scope
of the invention.